Culture medium for in vitro transport and storage of cells

Abstract

The present invention relates to an in vitro storage and / or transport of cells, comprising: - contacting cells with an aqueous thermoreversible gelation composition, which composition is in liquid form and comprises: (A) a thermoreversible gelation polysaccharide selected from the group consisting of carrageenan, gellan gum and konjac gum; and (B) carboxymethylcellulose as a thickening agent; - thermoreversible gelation of the composition by lowering the temperature; - storage and / or transport of the in vitro cultured cells embedded in the obtained hydrogel. The present invention also relates to a specific thermoreversible gelation composition for use in this method.

Description

Technical Field

[0001] This invention relates to the in vitro transport and storage of cells, and more particularly to gelled compositions for use as suitable culture media for culturing and / or isolating cells during transport or storage operations. Background Technology

[0002] When transporting in vitro cultured or isolated cells, the cells must be preserved in an appropriate culture medium to maintain their integrity and viability during operations such as long-distance transport and / or intermediate storage.

[0003] Methods for transporting cells in frozen flasks containing dry ice or liquid nitrogen have been described and are commonly used; however, this refrigerated transport is costly in addition to the problems caused by the hazards of dry ice and liquid nitrogen. Furthermore, these methods require cells to undergo a long acclimatization period (typically several days or weeks) after transport before they can be used.

[0004] Alternatively, cells can be transported in a culture state, typically by surrounding adherent or freely suspended cells with liquid culture medium. However, extreme care must be taken with transport in cases that may significantly affect cell integrity and / or cell functional capabilities (such as cell adhesion).

[0005] Maintaining the integrity and functional capacity of cells is particularly important for in vitro cultured cells used as models (e.g., for modeling genetic, biochemical, metabolic, or physiological processes), and more generally, for cells cultured on specific vectors, and especially for co-cultured cells placed on asymmetric vectors such as Transwell-type vectors. In such cases, it is essential not only to maintain the integrity of individual cells but also to preserve the overall structure of the culture, especially when the culture is intended for use as a model.

[0006] Methods have been developed to avoid the aforementioned problems, in which cultured cells are transported in a semi-solid culture medium that is typically a gel.

[0007] In this regard, particular reference can be made to the method described in patent US 8,900,842, which utilizes a gelatin-based gelling composition to store and / or transport cell cultures of in vitro tissues on an asymmetric carrier. In US 8,900,842, cell cultures of tissues are coated with a culture medium comprising gelatin, and then the gelatin is gelled at a temperature between 15°C and 25°C, allowing for transport / storage within the same temperature range. This method is particularly noteworthy because the resulting gelation is thermosensitive and reversible. That is, after transport / storage, liquefaction can be readily achieved simply by incubating the solidified composition, including the cells, at 37°C (which induces liquefaction of the gelatin). Liquefaction allows for washing of the composition used for transport / storage and replacement of the gelatin with the culture medium.

[0008] Other methods for using gels to transport / store cells have been described, but they are generally less noteworthy because these gels are not directly reversible for cell recovery.

[0009] For example, the method in patent US 10,655,120 utilizes hydrogels, typically irreversible alginate-based hydrogels, which require chemical decomposition or dissolution, which can be detrimental to the cell and cell culture structure in the case of complex cultures such as models or co-cultured cells.

[0010] Agarose-based gel methods are also being considered. These gels are thermosensitive and reversible, but liquefaction (the gel-sol process) means that high temperatures (typically at least 50°C) are incompatible with cell preservation. A specific method is described in US 8,709,803, in which agarose is used in a mixture of agarases that are inactivated at low temperatures (25°C or lower, where the mixture of agarose and agarase, once solidified, is capable of cell transport) and reactivated by incubation at 37°C, which causes the agarose to undergo enzymatic digestion of the gel and induces its liquefaction. Additional compounds are also required in this method for cell recovery.

[0011] Existing methods using thermosensitive reversible gels, particularly the method using gelatin disclosed in US 8,900,842 or the agarose-agarase mixture used in US 8,709,803, have the problem that these methods require time in both the formation of the gel before transport / storage (“sol-gel time”) and the liquefaction of the gel after transport / storage (“gel-sol time”). The duration of each of the sol-gel and gel-sol steps is typically several hours. Summary of the Invention

[0012] Purpose of the invention

[0013] One object of the present invention is to provide a method for in vitro transport and / or storage of cells that has the advantages of existing methods utilizing thermosensitive reversible gels, but requires only a short working time to form the gel and subsequently recover the cells. In particular, the working time required by the present invention is shorter than the total duration of the sol-gel step and gel-sol step in US 8,900,842 and US 8,709,803.

[0014] In this regard, the present invention is particularly intended to provide a method suitable for transporting or storing cells cultured or co-cultured on asymmetric carriers such as Transwell-type carriers, and more broadly suitable for cell cultures with specific structures, such as cell cultures intended for use as models. Summary of the Invention

[0015] This invention utilizes a specific thermosensitive reversible gelation composition, referred to herein as "Composition C". The inventors have now discovered that, among other advantages described below, Composition C, in the same manner as compositions based on gelatin, for example, disclosed in US 8,900,842, but with a shorter total duration of the sol / gel and gel / sol steps, particularly makes cells suitable for in vitro transport and / or storage.

[0016] More specifically, one subject of the present invention is a method for storing and / or transporting cells (typically cultured cells or isolated cells, and preferably cultured cells) in vitro, comprising the following sequential steps:

[0017] ■ Step 1 : To contact cells with an aqueous, thermosensitive, reversible gelation composition C, said composition C being in liquid form and contained in an aqueous culture medium:

[0018] (A) A thermosensitive, reversible gelatinized polysaccharide selected from carrageenan, gellan gum, and konjac gum; and

[0019] (B) Carboxymethyl cellulose as a thickener;

[0020] ■ Step 2 : By lowering the temperature, the composition C obtained in step 1 that is in contact with cells is thermosensitively gelled, thereby obtaining a hydrogel for embedding cells;

[0021] ■ Step 3 : Storage and / or transportation of cells embedded in hydrogel obtained in step 2.

[0022] In most cases, the method of the present invention further includes the following additional step 4 after step 3:

[0023] ■ Step 4The cells in the hydrogel are released by liquefying the hydrogel by raising the temperature.

[0024] According to another aspect, a particular subject of the present invention is composition C for use in the above-described method, which comprises:

[0025] (A) A thermosensitive, reversible gelatinized polysaccharide selected from carrageenan, gellan gum, and konjac gum; and

[0026] (B) Carboxymethyl cellulose as a thickener. Detailed Implementation

[0027] Specifically, composition C according to the invention is based on an aqueous culture medium, which is typically a homogeneous water-based medium that may contain additives such as salts or water-soluble solvents. Based on the total weight of composition C, the aqueous culture medium of composition C typically contains at least 90% by weight of water. Most commonly, based on the total weight of composition C, the aqueous culture medium contains at least 95% by weight, preferably at least 97% by weight, and especially at least 98% by weight of water.

[0028] Typically, the aqueous culture medium of composition C is suitable for storing and / or transporting cells. An advantage of the method of the present invention is that almost any type of aqueous culture medium can be used as the aqueous culture medium for composition C. The ratio A / B can be adjusted depending on the nature and concentration of the salts present in the culture medium, and the presence of EDTA should be preferred, but there are no technical limitations on the culture medium in composition C.

[0029] Typically, before performing step 1 of the invention on cells to be transported and / or stored according to the invention, the cells are embedded in or coated with a culture medium, referred to herein as the "initial culture medium". In most cases, even if not strictly required according to the invention, the method of the invention advantageously includes a culture medium replacement step, wherein all or part of the initial culture medium is replaced with composition C. In this case, step 1 can usually be performed by removing all or part (and usually substantially all) of the initial culture medium and then adding composition C according to the invention. In this case, the composition of the aqueous culture medium of composition C can generally be the same as that of the initial culture medium, but the composition in composition C can also be different depending on possible variant forms.

[0030] The composition C according to the invention comprises a thermosensitive reversible gelling polysaccharide (A) as a major component, selected from carrageenan, gellan gum, and konjac gum. This compound (A) particularly imparts suitable elasticity to the hydrogel formed in step 2 and used in step 3.

[0031] According to one embodiment, compound (A) present in composition C according to the invention is carrageenan. An alternative embodiment utilizes gellan gum, and another embodiment uses konjac gum.

[0032] According to a particularly suitable embodiment, compound (A) comprises (and preferably) carrageenan, preferably κ-carrageenan with a viscosity preferably between 5 mPa·s and 25 mPa·s at 25°C and 0.3 wt% in water. The carrageenan useful according to the invention typically has a solubility in hot water of about 5 mg / mL. Suitable κ-carrageenan according to the invention is, for example, κ-carrageenan from red algae available from Sigma-Aldrich (corresponding CAS number 11114-20-8), or commercially available Grinsted, CW series from DuPont.

[0033] The second component of composition C according to the invention is a thickener (B), which is carboxymethyl cellulose. This second compound (B) imparts suitable stiffness to the hydrogel formed in step 2 and used in step 3, preventing any leakage during cell transport and / or storage. The combination of the elasticity of compound (A) and the stiffness imparted by component (B) enables the transport of virtually any type of cell, even cell cultures with complex structures such as those co-cultured on asymmetric carriers, while appropriately preserving both the cells and their characteristics, and also appropriately maintaining the organization of the cells within the culture structure, thus enabling long-distance transport.

[0034] Preferably, the compound (B) used according to the invention is carboxymethyl cellulose with a viscosity between 50 mPa·s and 200 mPa·s at 25°C and 4% by weight in water, and a solubility in cold water typically of about 40 mg / mL. Preferably, the carboxymethyl cellulose useful according to the invention has a viscosity of less than 40 mPa·s at 25°C and 2% by weight in water. Suitable carboxymethyl cellulose includes, for example, the following commercially available products:

[0035] - Sodium carboxymethyl cellulose from Fisher Scientific (CAS number 9004-32-4), for example, low viscosity USP, Spectrum. TM Or low-viscosity sodium carboxymethyl cellulose from Sigma-Aldrich;

[0036] - Acqualon CMC 7L2 or Acqualon CMC 7M2F from DuPont.

[0037] In particular, in order to obtain suitable elasticity and stiffness, it is generally preferred that the ratio of the total mass of compound (A) to the total mass of compound (B) in composition C according to the invention is between 15:85 and 40:60, preferably between 20:80 and 40:60.

[0038] Furthermore, in the composition C used according to the present invention, the total concentration of compounds (A) and (B), i.e., the ratio of the sum of the mass of compound (A) and the mass of compound (B) to the total volume of composition (C), is preferably between 6 g / L and 10 g / L, for example, preferably between 8 g / L and 10 g / L.

[0039] Typically, in composition C according to the invention, the concentration of compound (A) is from 1.5 g / L to 4 g / L, more preferably from 1.5 g / L to 2.3 g / L. Furthermore, the concentration of compound (B) is typically from 6 g / L to 8.5 g / L, more preferably from 7.7 g / L to 8.5 g / L.

[0040] According to one possible embodiment, the composition (C) according to the invention may further comprise ethylenediaminetetraacetic acid (EDTA) as a chelating agent. This chelating agent is particularly useful in assisting the degelatination process in step 4 by chelating excess, optionally derived from physiological cellular metabolism, during transport / storage; otherwise, aggregation would occur, especially when compound (A) is κ-carrageenan. When EDTA is used, the EDTA content in composition (C) is preferably less than 1 mM.

[0041] An attractive aspect of the invention is that the compounds required to obtain the results sought within the scope of the invention are not of animal origin. According to a particularly preferred embodiment of the invention, composition C does not contain any substance of animal origin.

[0042] Compared to the method described in US 8,900,842 that utilizes gelatin such as gelatin from pigskin, the possibility of providing a gelling composition free of animal origin is another significant advantage of the present invention.

[0043] First, this achieves ethical improvements and enables the attainment of, for example, the so-called 3Rs principle (reducing, optimizing, and replacing the use of animals in scientific research). The in vitro transport and storage method of this invention represents a genuine improvement in this regard and avoids animal-derived products that in vitro systems typically still heavily rely on. In particular, this invention avoids the presence of the following animal-derived components (preferably not present in the composition of the compositions used in this invention): fetal bovine serum (FCS), animal-derived enzymes, collagen, and gelatin.

[0044] Furthermore, in addition to the aforementioned ethical considerations, the possibility of providing an animal-free technical solution as proposed by this invention also offers a more impartial technical advantage. That is, the absence of animal-derived compounds reduces experimental variability related to animal use and different batches of the product, resulting in more controlled products and ensuring optimal reproducibility and quality for downstream applications. More importantly, the absence of animal-derived compounds reduces the risk of contamination, especially when compared to the use of gelatin.

[0045] When needed, composition C of the present invention can be advantageously used in a global process that avoids the use of any animal-derived compounds, since it is able to avoid the use of any animal-derived compounds. In particular, according to one embodiment, composition C of the present invention does not contain serum, such as fetal bovine serum, and is not used with such serum.

[0046] In this regard, the culture medium used as the aqueous culture medium for composition C can be, for example, selected from the following serum-free culture media:

[0047] -PneumaCult from STEMCELL Technologies TM -ALI culture medium and supplements

[0048] -PneumaCult from STEMCELL Technologies TM -Ex culture medium and supplements

[0049] - StemSpan SFEM from STEMCELL Technologies

[0050] -STEMdiff from STEMCELL Technologies TM APEL TM culture medium

[0051] - X-VIVO 15 from Lonza Bioscience

[0052] - XVivo 10 from Lonza Bioscience

[0053] -CellGro DC from Corning

[0054] - CellGenix GMP DC from CellGenix, serum-free

[0055] - CD-U3 supplement from Biochrom

[0056] -Small Airway Epithelial Cell Growth Medium from PromoCell

[0057] -MP-hybridoma medium from MP Biomedicals

[0058] - Human Airway Epithelial Cell (hAEC) Culture Medium from Epithelix

[0059] -SmallAir medium from Epithelix

[0060] - MucilAir from Epithelix TM Culture medium.

[0061] Furthermore, according to a preferred embodiment, the method of the present invention is advantageously carried out without the addition of serum. Non-animal-derived serum substitutes can be used (when one of the culture media listed in the foregoing paragraphs is used as an aqueous culture medium, a serum substitute is not required), including, for example, the following:

[0062] -From Gibco TM of serum alternatives

[0063] -Cellastim InVitria from Bioscience

[0064] -From FUJIFILM Wako Chemical Corporation serum alternatives

[0065] - Artificial serum from Funakoshi that is xeno-free or animal-free.

[0066] - Serum Replacement Solution from PeproTech

[0067] -MITO+ Serum Extender from Corning

[0068] More generally, the objective of the invention can be achieved by using compounds A, B, and optionally EDTA alone in an aqueous culture medium. Alternatively, composition C according to the invention can advantageously omit other compounds.

[0069] In particular, according to a preferred embodiment, composition C does not contain all or part of the following compounds (and preferably composition C does not contain any of the following compounds):

[0070] - Gelatin, such as GPS (gelatin derived from pigskin), results in longer gelation and degelation times;

[0071] - Alginate and / or pectin (interact with multivalent ions, thus hindering thermosensitive reversibility);

[0072] - Agarose or agar (which causes the temperature increase required for the hydrogel to become thermosensitive and reversible);

[0073] -Enzymes;

[0074] - Serum, such as FCS;

[0075] -antibiotic.

[0076] The useful composition C according to the invention has another advantage due to its composition: since compounds (A) and (B) and optional EDTA are very readily soluble in water, composition C of the invention can be prepared very simply and quickly by dissolving compounds (A) and (B) plus optional EDTA in an aqueous phase, for example by introducing the compound in powder form into an aqueous culture medium heated to 50°C to 60°C (and then cooling the resulting composition C before contacting it with cells), complete and rapid dissolution can be obtained very easily.

[0077] Regardless of the exact composition of composition C, composition C according to the invention can be used in a very simple manner: in particular, obtaining the hydrogel (semi-solid composition) in step 2 and liquefaction (degellation) in step 4 are merely thermosensitive induced and do not require any chemical or enzymatic reactions.

[0078] More precisely, the hydrogel in step 2 is obtained by lowering the temperature below the gelation temperature of compound C, which is typically about 30°C.

[0079] Therefore, step 1 of the method is typically carried out at a temperature above 30°C, for example between 30°C and 60°C, and preferably between 30°C and 40°C, for example between 34°C and 39°C (typically about 37°C), in which composition C is used in liquid form. Step 2 is typically carried out by lowering the temperature to below 30°C, preferably below 20°C, for example between 1°C and 15°C (especially between 4°C and 10°C). Conversely, the liquefaction (degellation) in step 4 is obtained by heating the hydrogel to above its liquefaction temperature, which is also about 30°C. Therefore, step 4 of the method of the present invention is typically carried out by raising the temperature to above 30°C, preferably between 30°C and 40°C, for example between 34°C and 39°C (typically about 37°C).

[0080] Typically, using the specific composition C according to the invention, hydrogel formation (sol / gel transition) can be achieved rapidly in step 2, and liquefaction (gel / sol transition) is also relatively rapid in step 4, wherein the total duration of steps 2 plus step 4 is generally less than 4 hours, and in many cases less than 3 hours. This is a significant advantage compared to methods proposed in the prior art, especially compared to the method using gelatin disclosed in US 8,900,842 or the method using an agarose / agarosease mixture in US 8,709,803, because the total duration of the sol / gel plus gel / sol transition steps in these prior art methods is much longer (at least 5 hours).

[0081] According to an attractive implementation plan, step 2 can be performed in less than 90 minutes.

[0082] When the temperature used in step 2 is lower, the time required for hydrogel formation in step 2 is generally shorter, but this also depends on the starting temperature, i.e., the temperature used in step 1. With a lower temperature used in step 2 and the temperature in step 1 between 30°C and 40°C, hydrogel formation can typically occur within 30 to 60 minutes or even less, whereas the methods using US 8,900,842 or US 8,709,803 generally require at least approximately 2 to 3 hours.

[0083] Similarly, according to an attractive implementation, step 4 of the method of the present invention can be performed in less than 150 minutes.

[0084] When the temperature used in step 2 is lower, the time required for hydrogel formation in step 2 is generally shorter, but this also depends on the starting temperature, i.e., the temperature used in step 1. With a lower temperature used in step 2 and the temperature in step 1 between 30°C and 40°C, hydrogel formation can typically occur within 60 to 120 minutes or even less, whereas the methods using US 8,900,842 or US 8,709,803 generally require at least approximately 3 to 4 hours.

[0085] Given the favorable mechanical properties imparted by compounds (A) and (B), a longer transport / storage period for step 4 can be considered. Typically, step 4 can be carried out for at least 36 hours, which enables long-distance cell transport.

[0086] The method of the present invention can deliver any type of cell, including:

[0087] - Isolated or cultured cells (including single cultures and co-cultures)

[0088] - Free, non-adherent cells (in the first case, during the contact in step 1, the cells are typically embedded in composition C, which serves as the cell dispersion medium) or adherent cells on a carrier (in the second case, during the contact in step 1, the cells are typically coated with composition C). The carrier can be of any type, and in particular includes inserts, flasks, multi-walled plates, petri dishes, or other plastic containers.

[0089] According to a particularly attractive embodiment, the cells transported and / or stored according to the invention are co-cultured cells adhered to an asymmetric carrier, typically adhered to both sides of a Transwell nest.

[0090] The following examples correspond to this possible implementation scheme, thereby illustrating the present invention.

[0091] Example:

[0092] The following cell lines were used in this embodiment: A549, EA.hy926, THP-1, and Mφ-THP-1. Their characteristics are as follows:

[0093] - The A549 cell line corresponds to type II human alveolar epithelial cells capable of producing surfactant.

[0094] -EA.hy926 cell line is a somatic cell hybrid with endothelial characteristics;

[0095] -THP-1 is a human monocytic leukemia cell line; and

[0096] -Mφ-THP-1 are macrophages derived from THP-1 cells, differentiated using PMA (phorbol-12-myristate-13-acetate) or 1,25-dihydroxyvitamin D3.

[0097] Use these cells according to the following protocol:

[0098] 1) Cell seeding with asymmetric vectors

[0099] Cells were grown routinely in T75 flasks and triedpsinized twice a week.

[0100] Culture medium (in Transwell cell culture plates) TM (In nested cell culture flasks) Change the cells every other day. Maintain the cells in a humid atmosphere at 37°C with 5% CO2, and check for mycoplasma contamination regularly.

[0101] EA.hy 926 endothelial cells were seeded into inverted Transwell cells. TM Nested (1.2×10) 5 cells / cm 2 ; Pore size 1μm; 0.3cm 2 Once the cells have attached to the nested basal side, the plate is rotated back to its original orientation, and epithelial cells (A549) are seeded into the Transwell. TM The top of the membrane (0.83×10) 5 cells / cm 2 0.3cm 2 Epithelial and endothelial cells were grown at 37°C for three days, with 200 μL of culture medium on the apical side and 900 μL of culture medium on the basal side.

[0102] The preparation method of the inoculation medium used is as follows:

[0103] - Take 62.5 mL of DMEM (Dulbecco modified Eagle medium) from a new bottle (500 mL) and add 50 mL of FBS (10%), then add 12.5 mL of HEPES stock solution (1000 mM; sterile, filtered) to obtain HEPES buffered medium (25 mM).

[0104] The co-culture medium used was prepared as follows:

[0105] - Take 12.5 mL of DMEM medium from a new bottle (500 mL) and add 12.5 mL of HEPES stock solution (1000 mM; sterile, filtered) to obtain HEPES buffered medium (25 mM).

[0106] Mix and take out 125mL.

[0107] Add 50 mL of IMDM medium, then add 75 mL of RPMI+glutamine medium.

[0108] 2) Preparation of the gelling composition according to the present invention

[0109] κ-carrageenan from red algae (Sigma Aldrich) and carboxymethyl cellulose (sodium carboxymethyl cellulose from Sigma Aldrich - CAS No.: 9004-32-4, Product No.: C5678), referred to herein as "CMC," were dissolved in different concentrations as described in the following table in the co-medium defined according to step 1) to obtain five compositions C1-C5 according to the invention:

[0110]

[0111] Powdered κ-carrageenan and CMC were added to the culture medium while stirring at 60°C. The gelled composition was sterilized using a sterile vacuum filtration system (0.22 μm pore size) while still warm (>50°C).

[0112] They were then divided into appropriate volumes and supplemented with 10% serum substitute. The gelled composition was restored to 37°C before use according to the invention.

[0113] 3) Gel formation of second-generation cultures (according to steps 1 and 2 of the present invention)

[0114] From Transwell TM Remove the cell culture medium from both sides of the nested container and replace it with composition C1 as described in paragraph 2), and keep it in a water bath at 37°C until use (300 μL for the top side and 900 μL for the base side). Compositions C2 through C5 can be used in the same manner.

[0115] Cool the temperature between 17°C and 21°C for 60 minutes, keeping the perforated plate under the laminar flow hood open, then close and seal the plate.

[0116] 4) Transportation / storage of gelled second-generation cultures (according to step 3 of the invention)

[0117] The obtained gelled plates were placed in a refrigerated box at a temperature between 4°C and 10°C and transported for 24 hours.

[0118] 5) Degelatinization of second-generation cultures (according to step 4 of the present invention)

[0119] Remove the seal and place the gelled second-generation culture in an incubator (37°C, 5% CO2, 95% humidity) for 2 hours. Remove the liquefied composition by pipetting and wash the nested sides three times with cell culture medium to remove any potential gel residue.

[0120] 6) Assembly of third-generation cultures

[0121] Third-generation cultures were prepared from degelatinated second-generation cultures according to the following protocol:

[0122] Macrophage-like cells differentiated from THP-1 were isolated using PMA. Cells were prepared in cell culture medium at a density of 1.8 × 10⁶ cells / year. 5 A cell suspension of 1 cell / mL was prepared. After removing the gel, 200 μL of this suspension was placed on the top side of each nest, and 900 μL of culture medium was placed on the base side. After 4 hours, macrophages should have attached; the culture medium was then completely removed from the top side, and the culture medium on the base side was reduced to 200 μL (ALI production conditions).

Claims

1. A method for in vitro storage and / or transportation of cells, comprising the following consecutive steps: Step 1 contacting the cells with an aqueous temperature-sensitive reversible gelation composition C, the composition C being in liquid form and comprising in an aqueous medium:​ (A) a temperature-sensitive reversible gelation polysaccharide selected from carrageenan, gellan gum and konjac gum; and (B) carboxymethylcellulose as thickening agent Step 2 : thermally gelling the composition C in contact with the cells obtained in step 1 by lowering the temperature, thereby obtaining a hydrogel embedding the cells;​ Step 3 : storing and / or transporting the cells embedded in the hydrogel obtained in step 2.​ 2. The method according to claim 1, further comprising the following additional step 4 after step 3: Step 4 : liquefying the hydrogel by raising the temperature, thereby releasing the cells in the hydrogel.​ 3. The method according to claim 1 or 2, wherein the aqueous medium of composition C is a medium suitable for storage and / or transportation of cells.

4. The method according to claim 1, wherein step 1 is performed at a temperature between 30°C and 60°C.

5. The method according to claim 1, wherein step 1 is performed at a temperature between 30°C and 40°C.

6. The method according to claim 1, wherein step 2 is performed by lowering the temperature to below 30°C.

7. The method according to claim 1, wherein step 2 is performed at a temperature between 1°C and 15°C.

8. The method according to claim 2, wherein step 4 is performed at a temperature between 30°C and 40°C.

9. The method according to claim 2, wherein step 4 is performed at a temperature between 34°C and 39°C.

10. The method according to claim 1, wherein the total duration of step 2 plus step 4 is below 4 hours.

11. The method according to claim 1, wherein the total duration of step 2 plus step 4 is below 3 hours.

12. A composition C suitable for the method according to any one of claims 1 to 11, comprising in an aqueous medium: (A) a temperature-sensitive reversible gelation polysaccharide which is carrageenan; and (B) carboxymethylcellulose as thickening agent, wherein the ratio A / B of the total mass of compound (A) to the total mass of compound (B) is between 15:85 and 40:

60.

13. The composition C according to claim 12, wherein compound (A) comprises kappa-carrageenan.

14. The composition C according to claim 12, wherein compound (B) is carboxymethylcellulose having a viscosity below 40 mPa.s at 2% by weight in water at 25°C.

15. The composition C according to claim 12, wherein the ratio of the total concentration of compounds (A) and (B), i.e. the sum of the mass of compound (A) plus the mass of compound (B), to the total volume of composition (C) is between 6 g / L and 10 g / L.

16. The composition C according to any one of claims 12 to 15, further comprising ethylenediaminetetraacetic acid (EDTA) as chelating agent.

17. The composition C according to any one of claims 12 to 15, which does not comprise any substance from animal origin.

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